JPH10177023A - Method for examining metastasis of cancer and prognosis of patient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To examine the presence/absence of the metastasis of a cancer and estimate prognosis of a cancer patient by measuring the amount of a vascular endothelial cell growth factor vascular permeability factor(VPF) in human blood serum. SOLUTION: A monoclonal antibody and a polyclonal antibody to a human VPF 121 are used to measure the amount of a VPF according to an enzyme immunoassay. VFP amounts in blood serums of lung cancer patients with and without transfer are measured. Patients positive to metastasis show a significantly high value as compared with patients negative to metastasis, and therefore, the presence/absence of the metastasis can be detected from the VPF amounts. When the VPF amounts in the blood serum before and after a relapse of the lung cancer are measured, the VPF amount after the relapse is significantly high in comparison with before the relapse. Therefore, the measurement of the VPF amount in the blood serum can be utilized as a method for monitoring the relapse. The presence/absence of the metastasis of the cancer of a lung cancer patient can be examined and a prognosis can be estimated in this manner.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a vascular endothelial cell growth factor or a vascular permeability factor known as a factor that induces angiogenesis in the serum of a cancer patient.
endothelial growth factor or vascular permeability
factor, hereinafter abbreviated as VPF) by measuring the abundance of a factor called, to predict the presence or absence of cancer metastasis in the patient and predict the prognosis of the patient, that is, to predict the metastasis and prognosis of cancer in cancer patients. The present invention relates to a test method, and is effective as a test method useful for clinical diagnosis and determination of a treatment method, and is used in the pharmaceutical industry, particularly in the test drug industry and also in the biochemical test industry.

[0002]

2. Description of the Related Art In treating cancer patients, it is naturally important to treat cancer at the primary site, but even when the primary site has been completely cured, attention must be paid to the metastasis and recurrence of the cancer. It is also important to find metastases and recurrences as soon as possible and to treat these metastases and recurrent cancers without delay. Among lung cancers, lung CEA has been suggested that serum CEA level is a prognostic factor, but negative results have been reported and have not been widely used. In addition, a marker useful for detecting the presence or absence of metastasis is not currently reported in any cancer.

[0003] On the other hand, it is widely used to use a trace amount of a physiologically active substance present in a living body, that is, a substance exhibiting a pharmacological action in a very small amount, as a diagnostic marker, an indicator for judging a therapeutic effect or monitoring after surgery. And also angiogenesis, the proliferation of capillary endothelial cells,
The phenomenon of migration and tissue infiltration is known to play an important role in physiological or pathological phenomena such as fetal growth, wound healing, and cancer cell proliferation [(Fo
lkman, J., Cancer Res. 46: 467 (1986)]. As factors that induce angiogenesis, basic fibroblast growth factor (basic fibroblast growth factor) is a substance that directly acts on vascular endothelial cells.
growth factor, bFGF), acid fibroblast growth factor (acidi
c fibroblast growth factor, aFGF), vascular endothelial growth factor
factor / vascular permeability factor, VPF), platelet-derived endothelium
ial cell growth factor (PD-ECGF), etc.
wth factor-α (TGF-α), transforming growth factor
-β (TGF-β), angiogenin, tumor necrosis factor-α
(TNF-α) etc. [Folkman J. & Shing
Y., J. Biol. Chem., 267: 10931 (1992)].

[0004] Among them, VPF is secreted by normal or tumor cell lines of mouse, rat, guinea pig, bovine and human, and it is apparent that VPF is present in brain, pituitary gland, kidney and ovary by tissue. [(Ferrar
a N., et.al. Endocrine Reviews 13:18 (1992)], human VPF is angiogenesis and metastasis of breast cancer [Weider N., et.al.
Engl. J. Med. 324: 1 (1991)] and angiogenesis in renal cell carcinoma [Ayumi of Medicine, 168: 231 (1994)], or angiogenesis in retinal diseases [Adamis AP et.al., Biochem. Biophys. .
Res. Comm., 193: 631 (1993)].

[0005]

DISCLOSURE OF THE INVENTION The present inventors have proposed a method for examining the presence or absence of cancer metastasis and a method for predicting the prognosis of a cancer patient.
The possibility of using the method for measuring the amount of VPF in serum was examined.

[0006]

Means for Solving the Problems The present inventors used VPF enzyme immunoassay to measure serum VPF in cancer patients, particularly lung cancer patients.
The present inventors have found that by measuring the concentration, it is possible to examine the presence or absence of metastasis of cancer and predict the prognosis of a cancer patient. That is, the present invention relates to the following inventions. The first invention relates to a method for examining cancer metastasis and prognosis, which comprises measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum. The second invention relates to a method for examining metastasis and prognosis of cancer, wherein the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum is measured by a labeled immunoassay. The third invention is a cancer characterized in that the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum is measured using a substance that specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. Invention relating to a method for examining metastasis and prognosis. The fourth invention is to determine the abundance of vascular endothelial cell growth factor / vascular permeability factor in human serum using anti-vascular endothelial cell growth factor / vascular permeability factor antibody or vascular endothelial cell growth factor / vascular permeability factor receptor. An invention relating to a method for examining metastasis and prognosis of cancer, which is characterized by measuring. A fifth invention relates to an agent for examining cancer metastasis and prognosis, which comprises a substance that specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. The sixth invention relates to a drug for detecting metastasis and prognosis of cancer, which comprises an anti-vascular endothelial cell growth factor / vascular permeability factor antibody or a vascular endothelial cell growth factor / vascular permeability factor receptor.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The human VPF gene
The cDNA has already been isolated, the nucleotide sequence has been determined, and the amino acid sequence has been deduced. From this gene, four types of proteins differing in the number of amino acid residues (121
, 165, 189, and 206), and 121 amino acid residues (V
PF121) and 165 amino acid residues (VPF16
5) is said to be a mature protein [(Ferrara N., et.a
l. Endocrine Reviews 13:18 (1992)]. VPF121 is VP
Although the carboxyl-terminal 44 amino acids of F165 are deleted, it is not clear whether there is a difference between VPF121 and VPF165 in their effects on vascular endothelial cells. VPF acts by binding to the VPF receptor to promote cell proliferation and membrane permeability. As the VPF receptor, human VP
For F, FLT (M. Shibuya, et al, Oncogene, 5:
519 (1990)) and KDL (BI Terman et al, Bichem. Bio
phys. Res. Commun. 187: 1579 (1992)) are known.

[0008] On the other hand, a monoclonal antibody against human VPF121 has already been obtained by the present inventors, and the VPF is determined by enzyme immunoassay using the monoclonal antibody and a polyclonal antibody against human VPF121.
Can be measured (JP-A-8-16)
9898: Peptides and monoclonal antibodies). In addition, the present inventors have already established a method for detecting VPF in an enzyme immunoassay, and a Vpg of several pg / ml has been established.
It has been shown that PF can be detected (Japanese Patent Application No.
141271: Method for measuring vascular permeability factor). As a method for measuring the amount of vascular endothelial cell growth factor / vascular permeability factor,
In addition to the enzyme immunoassay described above, erythrocytes, latex, radioisotopes, enzymes, luminescent substances, fluorescent substances,
A labeled immunoassay using a metal molecule, a metal gel, a bacteriophage, or the like can be used.

[0009]

【Example】

(1) Preparation of anti-VPF polyclonal antibody The isolated human VPF cDNA was produced in Escherichia coli as a fusion protein (GST-VPF) with glutathione S-transferase (GST), and the resulting protein was used as an antigen to prepare rabbit anti-VPF according to a conventional method. Polyclonal antibodies were made.
The serum of the rabbit with an increased antibody titer was separated, and an IgG fraction of a rabbit anti-VPF polyclonal antibody was obtained by anion exchange column chromatography.

(2) Enzyme Labeling of Anti-VPF Polyclonal Antibody A part of the IgG fraction was digested with pepsin to prepare F (ab ') 2, which was then bound to peroxidase (horseradish) by the hinge method and labeled with peroxidase. A rabbit anti-VPF polyclonal antibody was obtained.

(3) Measurement of Serum VPF Concentration in Lung Cancer Patients Serum VPF concentration in lung cancer patients was measured by an enzyme immunoassay as shown below. An anti-VPF polyclonal antibody (5 μg / ml) was seeded at 100 μl / well on a 96-well plate, allowed to stand at 4 ° C. overnight, and washed four times with 0.1% bovine serum albumin (BSA) and PBS. 1% BSA, 0.1
1M sodium chloride, 0.1% sodium azide, 0.1
Blocking with M sodium carbonate buffer pH 6.5 (37
After 4 hours at 1 ° C.), 1% BSA, 0.4% gelatin, 1%
50 mM sodium phosphate buffer, pH 7.4 containing 20 mM magnesium chloride, 20 mM sodium ethylenediaminetetraacetate, 0.1 M sodium chloride, 0.1% sodium azide.
Serum diluted to 3 with 0 (sample diluent) or standard VPF dissolved in the same sample diluent was added and left at room temperature for 1 hour.
After washing 6 times with 0.1% BSA and PBS, a peroxidase-labeled anti-VPF polyclonal antibody was added at 100 μl / well.
The reaction was continued for 1 hour at room temperature. Again, 0.1% BS
A. After washing 8 times with PBS, 0.125% (w / v) orthophenylenediamine, 0.015% hydrogen peroxide, 0.2M tris (hydroxymethyl (aminomethane) -citrate buffer (p
H5.2) was added in an amount of 100 μl / well, and reacted at room temperature for 30 minutes. 2N sulfuric acid was added in an amount of 100 μl / well, and the reaction was stopped.
Plate absorbance (M-Vmax, Molecular Devic
es).

(4) Measurement of Serum VPF Concentration in Lung Cancer Patients Among the lung cancer patients, 19 patients with metastasis (metastasis-positive patients) and patients without metastasis (metastasis-negative patients) 35
For the examples, the amount of serum VPF was measured. Table 1 shows the results of calculating the average value and the standard deviation value of the measured values of each patient. Metastasis positive patients showed significantly higher values than metastasis negative patients. Therefore, it became clear that the presence or absence of metastasis can be determined by measuring the amount of VPF in serum. In addition, the serum VPF levels before and after recurrence of four lung cancer patients were measured, and changes at the individual level are shown in FIG. Furthermore, the average value and standard deviation value of four cases before and after recurrence were calculated (Table 1). As is apparent from these results, the VPF level after recurrence was significantly higher than that before recurrence, indicating that measurement of serum VPF level can be used as a method for monitoring recurrence. Became.

[0013]

[Table 1] Note: *, ** means p <0.05.

[0014]

According to the present invention, there is an excellent effect that the presence or absence of metastasis of cancer in a cancer patient, particularly a lung cancer patient, and the prognosis of the cancer patient can be easily predicted.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of measuring the VPF concentration in the serum of a lung cancer patient before and after recurrence.

 ──────────────────────────────────────────────────続 き Continued on front page (72) Inventor Katsuhiko Matsuo 2nd Okubo Tsukuba, Ibaraki Pref.

Claims (6)

[Claims] 1. A method for examining metastasis and prognosis of cancer, which comprises measuring the amount of vascular endothelial cell growth factor / vascular permeability factor in human serum. 2. The method according to claim 1, wherein the method for measuring the abundance of vascular endothelial cell growth factor / vascular permeability factor is a labeled immunoassay. 3. The method according to claim 1, wherein the abundance of vascular endothelial cell growth factor / vascular permeability factor is measured using a substance that specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. Of cancer metastasis and prognosis. 4. The substance which specifically reacts with vascular endothelial cell growth factor / vascular permeability factor is an anti-vascular endothelial cell growth factor / vascular permeability factor antibody or a vascular endothelial cell growth factor / vascular permeability factor receptor. 4. The method for examining metastasis and prognosis of cancer according to claim 3, wherein: A method for examining the presence or absence of cancer metastasis. 5. An agent for testing cancer metastasis and prognosis, comprising a substance which specifically reacts with vascular endothelial cell growth factor / vascular permeability factor. 6. A substance which specifically reacts with vascular endothelial cell growth factor / vascular permeability factor is an anti-vascular endothelial cell growth factor / vascular permeability factor antibody or a vascular endothelial cell growth factor / vascular permeability factor receptor. The cancer metastasis and prognosis test agent according to claim 5, characterized in that: